Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
  • C07D498/08—Bridged systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D213/79—Acids; Esters
  • C07D213/80—Acids; Esters in position 3
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents

Definitions

• This invention relates to novel rifamycin antibiotic derivatives of general formula I: ##STR2## and to the oxidated derivatives thereof of formula Ia: ##STR3## wherein: R represents, halo, hydroxy, thio, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alkylthio, (C 1 -C 4 )acyloxy, (C 1 -C 4 )alkylamino, di(C 1 -C 4 )alkylamino or a group of formula: ##STR4## wherein: R 3 represents (C 1 -C 4 )alkyl or (C 3 -C 6 )cycloalkyl;
• R 4 represents a group of formula ##STR5## wherein: R 6 and R 7 independently represent hydrogen or (C 1 -C 4 )alkyl or
• R 6 and R 7 together with the adjacent nitrogen atom form a five or six membered heterocyclic ring, optionally containing one further heteroatom selected from oxygen, nitrogen and sulfur, wherein one of the carbon or nitrogen atoms of the ring is optionally substituted by a (C 1 -C 4 )alkyl moiety;
• R 5 is hydrogen or halo
• R 4 represents hydrogen or halogen
• R 9 represents (C 1 -C 4 )alkyl, or a six membered heterocycle ring containing one or two nitrogen atoms, wherein the carbon and nitrogen atoms of the ring are optionally substituted with one or two (C 1 -C 4 )alkyl moieties;
• R 1 is hydroxy in formula I or oxygen formula Ia
• R 2 represents hydrogen, a five or six membered heterocyclic ring containing one or two heteroatoms selected from oxygen, nitrogen and sulfur, wherein one of the carbon or nitrogen atoms of the ring is optionally substituted by a (C 1 -C 4 )alkyl moiety, or a group of formula
• R 10 represents a six membered heterocycle ring containing one or two nitrogen atoms, wherein one of the carbon or nitrogen atoms of the ring is optionally substituted by (C 1 -C 4 )-alkyl or (C 5 -C 6 )cycloalkyl;
• R 1 and R 2 taken together form a group of formula ⁇ N--(CHR 11 )--X--, --NH--(CHR 11 )--X--, or --N ⁇ (CR 11 )--X--,
• X represents a sulfur atom or a --NH--group
• R 11 represents hydrogen, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkylamino or di(C 1 -C 4 )alkylamino;
• (C 1 -C 4 )alkyl represent a linear or branched hydrocarbon moiety containing carbon atoms respectively, such as:
• halo represents fluoro, chloro, bromo or ioda
• (C 1 -C 4 ) alkoxy represents a linear or branched ether moiety containing 1, 2, 3 or 4 carbon, such as:
• (C 1 -C 4 )acyloxy represents a carboxylic moiety containing 1 to 4 carbon atoms, such as:
• (C 1 -C 4 )alkylthio represents a linear or branched thioether moiety with 1 to 4 carbon atoms, such as:
• (C 1 -C 4 )alkylamino represents an amino moiety substituted with a linear or branched alkyl containing 1 to 4 carbon atoms, such as:
• (C 1 -C 4 ) dialkylamino represents an amino moiety substituted with two linear or branched alkyl moieties containing 1 to 4 carbon atoms such as:
• (C 3 -C 6 )cycloalkyl represent a cyclic hydrocarbon moiety containing from 3 to 6 carbon atoms such as:
• a five or six membered heterocyclic ring containing one or two heteroatoms selected from oxygen, nitrogen and sulfur is an heterocycle ring such as: ##STR7## wherein W represents hydrogen or the various possible substituents of the 5 or 6 membered heterocycle ring, according to the definitions set out above;
• a bifunctional alkylenic chain optionally containing 1 or 2 nitrogen atoms, according to the meaninings of R 2 and R 3 taken together, is a group which forms, with the two adjacent carbon atoms, a six membered aromatic heterocyclic ring, such as: ##STR8## wherein the substituents R 8 and R 9 has the same meanings as in formula I;
• a group of formula ⁇ N--(CHR 11 )--X--, --NH--(CHR 11 )--X--or --N ⁇ (CR 11 )--X--, is a group which forms, with the adjacent carbon atoms in position 3 and 4,an heterocycle ring such as; ##STR9## wherein R 11 is as above defined; obviously, the double bond between the nitrogen atom and the carbon atom in position 4 is only possible when the rifamycin is in the oxidated form.
• Di(C 1 -C 4 )alkylamino represents an amino moiety substituted with two linear or branched alkyl groups containing 1, 2, 3 or 4 carbon atoms such as: --N--(CH 3 ) 2 , --N(CH 3 )(CH 2 --CH 3 ), --N(CH 2 --CH 3 ) 2 , --N(CH 3 )(CH 2 --CH 2 --CH 3 ), --N(CH 2 --CH 3 )(CH 2 --CH 2 --CH 3 ), --N(CH 2 --CH 3 )(CH 2 --CH 2 --CH 3 ), --N(CH 2 --CH 2 --CH 3 ) 2 , --N(CH 3 ) CH--(CH 3 ) 2 !, --N(CH 2 --CH 3 ) CH--(CH 3 ) 2 !, --N(CH 3 )(CH 2 --CH 2 --CH 3 ), --N( CH 2 --CH 3 )(CH 2 --CH 2 --CH 3 ), --N( CH 2 --CH 3 )(
• Pharmaceutically acceptable base addition salts of the compounds of formula I are the rifamycin salts formed with alkali metal, earth-alkali metal, (C 1 -C 4 )alkylamines, (C 1 -C 4 )alkanolamines or basic aminoacids.
• the hydroxy groups linked in positions 1 and 4 on the naphtalenic ring of the compound of formula I may be both in the reduced form (in such case R 1 is hydroxy) or oxidated form (R 1 is oxo in this case).
• rifamycin SV derivatives of rifamycin SV and of rifamycin S, respectively.
• the conversion from one form of rifamycin to the other, and viceversa, is easily carried out by means of oxidating or reducing reactions well known in the art; for instance the oxidation reaction may be carried out with manganese dioxide or potassium hexacyanoferrate(III) in chloroform, while the reducing reaction with ascorbic acid or sodium ascorbate in an hydroalcoholic solution.
• the hydroxy moiety in position 1 is also in the oxidated form when the substituent R 1 together with R 2 (in the compound of formula I) form a group of formula ⁇ N--(CHR 11 )--X--, wherein X and R 11 are as above defined; in this case the formation of the oxo moiety in 1 is a consequence of the double bonding between the nitrogen atom and the carbon atom in position 4.
• rifamycins is intended to comprise within its meanings all the suitable rifamycin and rifamycin-like compounds known in the art, such as rifamycin S. SV, P, the 3- and/or 4-derivatives thereof, and the pharmaceutically acceptable salts thereof.
• the rifamycin antibiotic compounds are well known in the art, as being widely used for long time in the treatment of infections caused by Mycobacteria and Gram positive microorganisms and prophylaxis for certain Gram negative infections.
• the best known member of this antibiotic family is Rifampicin, which is one of the antibiotics of choice in the treatment of tuberculosis, whose main causative agent is Mycobacterium tuberculosis.
• Rifamycin SV corresponds to the compound of formula I wherein the substituent R at position 36 is replaced by a hydrogen atom, R 2 is hydrogen and R 1 is hydroxy; rifamycin S is the oxidated form of rifamycin SV, as stated above; rifamycin P is the 4-desoxy-thiazolo- 5,4-c! rifamycin S; rifampicin is the 3- ⁇ (4-methyl-1-piperazinyl)imino!-methyl ⁇ rifamycin SV.
• rifamycin SV may be obtained by fermenting variant cultures of the strain ATCC 13685 Nocardia mediterranei (previously named Streptomyces mediterranei, now renamed as Amycolatopsis mediterranei); for instance from Nocardia mediterranei ATCC 21271, as described in "The Journal of Antibiotics vol. 22, 12, 637, (1969)".
• U.S. Pat. No. 3,884,763 discloses a process for preparing rifamycin SV or rifamycin S by aerobically fermenting an aqueous nutrient medium containing a strain of Micromonospora chalcea ATCC 21994.
• Rifamycin S and SV may also be obtained by chemical modification of the rifamycin B as described in U.S. Pat. No. 3,301,753.
• Rifamycin B was first obtained as a component of a rifamycin complex by fermenting the Streptomyces mediterranei strain ATCC 13685 as described in U.S. Pat. No. 3,150,046; rifamycin B may also be obtained as a single component by adding sodium ethyl barbiturate to the culture medium of Streptomyces mediterranei strain ATCC 13685, as described in U.S. Pat. No. 2,988,490 or by fermenting a variant culture of the strain ATCC 13685, i.e. the strain ATCC 21796, as described in U.S. Pat. No. 3,871,965.
• Rifamycin P may be obtained either by fermentation of Streptomyces mediterranei ATCC 31064, ATCC 31065, ATCC 31066, as disclosed in U.S. Pat. No. 4,263,404, or by chemical modification of rifamycin S, as described in U.S. Pat. No. 4,144,234.
• U.S. Pat. No. 4,880,789 discloses the preparation of the 2'-N,N-dialkylamino derivatives of rifamycin P, by treating rifamycin P with dialkylamine in ethyl acetate; such derivatives may also be obtained by treating the 3-bromorifamycin S with N,N-dialkylthiourea, as described by "Cavalleri B. et al., J. of Med. Chem., 1990, 33, 1470-1476".
• Rifampicin which is a rifamycin SV bearing a (4-methyl-1-piperazinyl)-imino!methyl group in position 3, may be obtained by reacting rifamycin SV with N-methylene-t-butylamine (obtained by reacting formaldehyde with t-butylamine) in the presence of manganese dioxide and then with 1-amino-4-methyl piperazine, as described in U.S. Pat. No. 3,542,762.
• U.S. Pat. No. 3,301,753 discloses the alkali and earth-alkali metal salts of rifamycin SV;
• U.S. Pat. No. 4,312,866 discloses the preparation of rifamycin SV salts with basic aminoacids, such as arginine, lysine and histidine.
• 25-O-deacetyl-25-O-propionyl and 25-O-deacetyl-25-O-pyvaloyl derivatives of rifamycin are described by Kump W. et al., Helv. Chem. Acta, 1973, 56, 2323.
• new antibiotic derivatives of rifamycin are provided which are mainly active against gram positive bacteria and gram positive as well as gram negative anaerobes, and show anti-microbial activity against rifamycin resistant strains.
• Preferred compounds of formula I are those compounds wherein:
• R is, halo, hydroxy, (C 1 -C 4 )acyloxy, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alkylthio, di(C 1 -C 4 )alkylamino or a group of formula: ##STR10## wherein: R 3 represents (C 1 -C 4 )alkyl or (C 3 -C 6 )cycloalkyl;
• R 4 represents a group of formula ##STR11## wherein R 12 represents hydrogen or (C 1 -C 4 )alkyl; R 5 is hydrogen or halo;
• R 4 together with R 5 form a bifunctional alkylenic chain, optionally containing 1 or 2 nitrogen atoms, of the following formula: ##STR12## wherein R 9 represents (C 1 -C 4 )alkyl, a group of formula, ##STR13## wherein R 13 is hydrogen or (C 1 -C 4 )alkyl, or a group of formula ##STR14## wherein R 14 and R 15 independently represent hydrogen or (C 1 -C 4 )alkyl;
• R 1 is hydroxy in the reduced form or oxygen in the oxydated form
• R 2 represents hydrogen, a six membered heterocyclic ring containing one or two heteroatoms selected from oxygen, nitrogen and sulfur, wherein one of the carbon or nitrogen atoms of the ring is optionally substituted by a (C 1 -C 4 )alkyl moiety, or a group of formula: ##STR15## wherein R 16 represents (C 1 -C 4 )alkyl or (C 5 -C 6 )cycloalkyl;
• R 1 and R 2 taken together form a group of formula --N ⁇ CR 11 --S--wherein R 11 represents hydrogen, (C 1 -C 4 )alkyl or di(C 1 -C 4 )alkylamino.
• R is propyl, butyl, octyl, fluoro, bromo, chloro, iodo, hydroxy, formyl, acetyl, thiomethyl diethylamino or a group of formula: ##STR16## wherein: R 3 is ethyl or cyclopropyl, R 4 is 4-methyl-1-piperazinyl and R 5 is hydrogen;
• R 1 is hydroxy in the reduced form or oxygen in the oxydated form
• R 2 is hydrogen, 4-morpholinyl, ⁇ (4-methyl-1-piperazinyl)imino!methyl ⁇ or ⁇ (4-cyclopentyl-1-piperazinyl)imino!methyl ⁇ ;
• Particularly preferred compounds are those compounds of formula I wherein:
• R is bromo, chloro, iodo, hydroxy or a group of formula: ##STR19## wherein: R 3 is ethyl, R 4 is 4-methyl-1-piperazinyl and R 5 is hydrogen;
• R 1 is hydroxy in the reduced form or oxygen in the oxydated form and R 2 is hydrogen, 4-morpholinyl or ⁇ (4-methyl-1-piperazinyl)-imino!methyl ⁇ .
• the preferred pharmaceutically acceptable salts of the compounds of formula I are the rifamycin salts formed with alkali metal or basic aminoacids; most preferred are the salts with sodium, arginine, lysine or histidine.
• A-further object of the present invention is to provide a process for preparing the compounds of general formula I. With said process it is also possible to prepare the rifamycin derivatives of formula I wherein R is a (C 1 -C 8 )alkyl group. Thus, only when referring to the preparation method, the meaning of R will encompass also this further group.
• U.S. Pat. No.4,086,225 discloses the preparation of 4-desoxy-imidazolo 4,5-c! rifamycin derivatives from Rifamycin S;
• U.S. Pat. No.4,880,789 describes the preparation of 2'-dialkylamino derivatives of Rifamycin P.
• 36-substituted rifamycin S or SV may be obtained according to the present process while the desired substituents are introduced in the other positions of the molecule only afterwards. This procedure is preferably followed when said further substituents would prevent the normal course of reaction or could itself undergo unwanted chemical modifications during the preparation of the 36-derivatives according to the process of the present invention.
• the compounds of formula I may be obtained by reacting the corresponding 25-O-deacetylated rifamycin with a suitable malonic acid derivative in the presence of a condensing agent.
• the above rifamycin starting materials may not be reacted as such, but has to be protected on the positions 21 and 23, before reacting it.
• Such protection is performed according to the methods known in the art for protecting geminal hydroxy groups and results in a ciclyzation of the two oxygen atoms of the hydroxy functions in position 21 and 23.
• 25-O-deacetylrifamycin S cyclic-21,23-(1-methylethylidene acetal)--wherein R 1 is oxo and R 2 is hydrogen-- may be prepared by reacting rifamycin S with acetone and anhydrous cupric sulfate or with 2,2-dimethoxypropane and sulfuric acid, and then hydrolyzing with NaOH, according to "W. Kump and H. Birchel, Helv.Chim.
• the protected rifamycin P may be prepared by reacting the 25-O-Deacetylrifamycin S cyclic-21,23-(1-methylethylidene acetal) with N-bromosuccinimide and 1,1-diethylthiourea in dimethylformamide, thus obtaining the corresponding 25-O-Deacetylrifamycin P cyclic-21,23-(1-methyl-ethylidene acetal).
• the process of the present invention therefore comprises:
• one of the two malonic carboxylic moieties reacts with the hydroxy moiety in position 25 of the compound of formula II, thus forming an ester moiety in such position.
• the second carbon atom of this moiety is conventionally numbered as C 36 .
• This reaction is generally conducted in the presence of an inert organic solvent which do not unfavorably interfere with the reaction course and is capable of at least partially solubilizing the antibiotic material.
• Said inert organic solvents are those commonly used in the art and comprise alkylamides, alkylnitriles, saturated linear or cyclic ethers, glycol ethers, phosphoramides, sulfoxides, chlorinated solvents or mixtures thereof.
• Preferred inert organic solvents are: dimethylformamide, acetonitrile, dimethoxyethane, tetrahydrofuran (THF), hexamethyl-phosphoramide, dimethylsulfoxide, chloroform and dichloroethane or mixtures thereof; most preferred is tetrahydrofuran.
• the condensing agent may be any substance commonly used in the art for the esterification reactions selected from carboxydiimides, dialkylaminopyridines, carbonylimidazoles, triphenilphosphine in carbon tetrachloride, substituted dithiocarbonates and diphenylphosphorilazides.
• said condensating agents are: 4-dimethylaminopyridine, N,N'-carboyl-bis imidazole, S,S'-bis-1-(phenyl-1H-tetrazol-5-yl)-dithiocarbonate and 1,3-Dicyclohexylcarbodiimmide (DCC).
• Preferred condensing agents are carboxydiimmides derivatives, 1,3-Dicyclohexylcarbodiimmide being the most preferred one.
• the above reaction is preferably conducted at temperatures from about 0° C. to 35° C., while the reaction time may vary from 1 to 2 hours. More preferably, the mixture is reacted at 0° C. for about 15 minutes and then for about one hour at room temperature.
• the removal of the acetonidic moiety bridging positions 21 and 23 is performed under mild acidic conditions in the presence of an inert organic solvent, as above defined.
• the acids commonly used for the cleavage of such moiety can be here conveniently utilized; these acids have to be in diluted form, to avoid the demolition of the substrate.
• Suitable acids are mineral acids (e.g. hydrochloric acid, sulfuric acid) or organic sulfonic acids (e.g. p-toluensulfonic acid), the preferred one being sulfuric acid.
• the reaction is conducted between 30° C. and 50° C., while the reaction time may vary from 14 to 18 hours. It is generally preferred to carry it out at a temperature of about 40° C. for about 16 hours.
• Step c of the present process permits the removal of the free carboxy moiety linked to the C 36 ,for obtaining the corresponding compound of general formula 1.
• any salt or oxide or a mixture thereof containing the cuprous ion can be employed.
• these compounds are: Cu 2 O, Cu 2 S, CuCl, CuBr and Cu 2 SO 4 , most preferred being cuprous oxide.
• the inert organic solvent to be used should be capable of at least partially solubilizing the antibiotic material and should not unfavorably interfere with the reaction course.
• the skilled man would be able to choose the most appropriate solvent among those commonly used in the art. Suitable solvents are those previously listed and, among those, the preferred solvent for this reaction is acetonitrile.
• the reaction temperature is from 50° C. to 75° C., preferably from 60° C. to 70° C.
• the reaction time may vary depending on the nature of substituent on the malonic moiety. For the most reactive compounds it is about one hour, while for the less reactive ones it raises up to 20 hours.
• the halogen ion exchange reaction for obtaining the 36-iodo rifamycin derivatives is easily accomplished by contacting the corresponding 36-halo rifamycin derivative with a metal alkali iodide, preferably sodium iodide, in the presence of acetone, as commonly known in the art.
• a metal alkali iodide preferably sodium iodide
• Such reaction is generally conducted at room temperature for about 3 to 5 hours.
• R is (C 1 -C 4 )acyloxy
• the 36-chloro, -bromo or -iodo rifamycin derivative obtained as above described is contacted with a (C 1 -C 4 )acylate salt, in the presence of an inert organic solvent.
• the (C 1 -C 4 )acylate salt is preferably an alkali metal, most preferred is potassium, or a silver salt, while the inert organic solvent is selected among those listed above, preferably being anhydrous dimethylformamide.
• the reaction is generally conducted at room temperature for about 16 to 26 hours.
• R is (C 1 -C 4 )alkylamino or (C 1 -C 4 )dialkylamino
• the 36-chloro, -bromo or -iodo rifamycin derivative obtained as above described is contacted with the correspondent alkyl or dialkylamine in the presence of an inert organic solvent.
• the inert organic solvent is preferably tetrahydrofuran and the reaction is generally conducted at room temperature for about 2 to 6 hours.
• the hydrolisis is conducted in an hydroalcoholic solution under mild basic conditions, at room temperature, for about 10 to 16 hours.
• the reaction is performed with potassium bicarbonate in a water/methanol mixture (rate from 1 ⁇ 2 to 1 ⁇ 3 v/v).
• R is a group of formula ##STR24## the 36-chloro, -bromo or -iodo rifamycin derivative obtained as above described is contacted with a salt of a 4-oxo-3-pyridinyl carboxylic acid derivative of general formula IV ##STR25## wherein R 3 ,R 4 and R 5 are as defined in formula I, in the presence of an inert organic solvent.
• a litium, sodium, potassium or silver salt of the 4-oxo-3-pyridinyl carboxylic acid derivative is employed.
• the 4-oxo-3-pyridinyl carboxylic acid derivatives may be commercially available compounds such as Pefloxacin®, Nalidixic acid or N-methyl-pipemidic acid, known compounds such as those described in U.S. Pat. No.5,075,319, or new compounds derived from the compounds of general formula Va or Vb: ##STR26## wherein Hal represents chloro or bromo and Y is hydrogen or lower alkyl.
• the compounds of formula IVa and IVb may be prepared by saponification of the corresponding 4,6-di-halo-nicotinic acid or lower alkyl 4,6-di-halo-nicotinate, as disclosed in Israeli Patent 44327/2.
• the compounds of formula Va are preferably reacted first with an alkylating agent, so to obtain the corresponding N-(C 1 -C 4 )alkyl or N-(C 3 -C 6 )cycloalkyl derivatives; afterwards this N-alkylated compound is reacted with a suitable amine in order to substitute the halogen with the desired moiety of formula --NR 6 R 7 , wherein R 6 and R 7 are as defined in formula I.
• suitable starting materials for the process of the present invention may be prepared by reacting a N-alkyl derivative of a compound of general formula IVa with N-methylpiperazine.
• the inert organic solvent that may be used in the present process should be capable of at least partially solubilizing the antibiotic material and should not unfavourably interfere with the reaction course.
• the skilled man would be able to choose the most appropriate solvent among those commonly used in the art, partcularly in view of the teachings of the present disclosure.
• Suitable solvents are those listed before when dealing with reaction of the malonic acid derivative with rifamycin. Particularly preferred is dimethylformamide.
• the reaction is conducted generally at a temperature between 15° C. and 40° C.
• the reaction time may vary from 10 to 24 hours.
• the salt of the compond of formula IV is first added to the inert organic solvent and stirred at room temperature for about 30 minutes, optionally in the presence of an activated molecular sieve, such as Union Carbide type 4 ⁇ (FLUKA); afterwards, the suitable 36-chloro, -bromo or -iodo rifamycin derivative is added to the solution and the mixture is stirred for from 14 to 20 hours at room temperature.
• an activated molecular sieve such as Union Carbide type 4 ⁇ (FLUKA)
• Preferred 36-halo rifamycin derivatives which may be employed for the present process are the 36-iodo derivatives, whilst preferred carboxylic acid salt is potassium salt.
• the separation of the reaction products is preferably accomplished by means of extraction with water-immiscible organic solvents or by adding non-solvents.
• water-immiscible solvent as used in this application, is intended to have the meaning currently given in the art to this term and refers to solvents that at the conditions of use are slightly miscible or practically immiscible with water in a reasonably wide concentration range, suitable for the intended use.
• water-immiscible organic solvents that can be used in the extraction of the antibiotic substances of the invention from an aqueous phase are: the usual hydrocarbon solvents which may be linear, branched or cyclic such as hexane or cyclohexane; halogenated hydrocarbons such as chloroform, carbon tetrachloride, dichloromethane, dichloroethane, fluorobromoethane, dibromoethane, trichloropropane, chlorotrifluorooctane and the like; aromatic hydrocarbons such as benzene, toluene, xylene and the like; esters of at least four carbon atoms, such as ethyl acetate, propyl acetate, ethyl butyrate, and the like; alkanols of at least four carbon atoms which may be linear, branched or cyclic such as butanol, 1-pentanol, 2-pentan
• precipitating agents are petroleum ether and lower alkyl ethers, such as ethyl ether, propyl ether and butyl ether; the preferred one being petroleum ether.
• reaction products may be obtained by precipitation with non-solvents or by chromatographic techniques.
• Precipitating agents suitable for purification are those listed above.
• the chromatographic techniques suitable for purifying the reaction products of the present process are those commonly known in the art and comprise partition chromatography, reverse-phase partition chromatography, ion-exchange chromatography, flash chromatography, affinity chromatography, HPLC techniques and the like. the preferred one being flash chromatography.
• the purification of the residue is accomplished by means of flash-chromatography; particulary preferred as stationary phase is silica gel, while preferred eluent is methanol in dichloroethane.
• the antimicrobial activity of the compounds of the present invention was demonstrated by a series of standard in vitro tests.
• the minimal inhibitory concentration (MIC) for the microorganisms was determined by broth microdilution methodology. Inocula were approximately 10 4 colony-forming units per ml (CFU/ml), except for Bacteroides fragilis, Clostridium perfringens and Propionibacterium acnes (10 5 CFU/ml).
• Incubation was at 37° C. Neisseria gonorrhoeae and Haemophilus influenzae were incubated in 5% carbon dioxide in air; C. perfringens, P. acnes and B. fragilis in nitrogen-carbon dioxide-hydrogen (80:10:10); other organisms in air. Incubation times were 48 hours for N. gonorrhoeae, H. influenzae, P. acnes and B. fragilis; 20-24 hours for other organisms.
• the growth media were: Iso-Sensitest broth (Oxoid) for staphylococci, Enterococcus faecalis, Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae, and Pseudomonas aeruginosa; Todd Hewitt broth (Difco) for streptococci; GC Base broth (Difco)+1% (v/v) IsoVitaleX (BBL) for N. gonorrhoeae; Brain Heart Infusion broth (Difco)+1% (v/v) Supplement C (Difco) for H. influenzae; Wilkins-Chalgren broth (Difco) for C. perfringens, P. acnes and B. fragilis.
• Compound C30 has further been tested against Mycobacterium tuberculosis and Mycobacterium avium, according to the following methodology:
• M. tuberculosis was grown for 2-3 weeks on Lowenstein-Jensen medium (Sclavo) and M. avium on 7H10 agar (Difco) for 2 weeks.
• the cultures were suspended in 7H9 broth (Difco), diluted in Becton Dickinson diluting fluid, and inoculated into Bactec 12B vials (Becton Dickinson).
• the inocula for control vials were 10 2 -10 3 cfu/ml; vials with compound C30 were inoculated with 100 times this number of cells.
• the vials were incubated at 37° C. and read daily in a Bactec 460 machine.
• M. tuberculosis was considered sensitive to a given concentration of antibiotic if the difference between growth indexes on successive days in the vial was less than that of the control.
• M. avium was considered sensitive to a given antibiotic concentration if the growth index was less than that of the control.
• the inhibitory concentration of compound C30 was found to be less than 0.2 against both the strains.
• mice were infected intraperitoneally with about 1 ⁇ 10 6 CFU of S. aureus Smith suspended in 0.5 ml of Difco bacteriological mucin.
• the compounds of the invention can be used as active ingredients in the preparation of medicaments for human or animal treatment.
• antibiotic compounds of general formula I are antimicrobial agents mainly active against gram positive bacteria, and fastidious gram negative bacteria.
• the compounds of the invention show a considerable antimicrobial activity against those strain which have developed resistance to rifampicin.
• the main therapeutic indication of the antibiotic substances of the invention is thus in the treatment of infections related to the presence of a microorganism susceptible to it.
• treatment is intended to encompass also prophylaxis, therapy and cure.
• the patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
• the compounds of the invention can be administered as such or in admixture with pharmaceutically acceptable carriers and can also be administered in conjunction with other antimicrobial agents generally employed in the art along with rifamycin antibiotics.
• Conjunctive therapy thus includes sequential, simultaneous or separate administration of the active compound in a way that the therapeutical effects of the first administered one has not entirely disappeared when the subsequent one is administered.
• the amount of active substance in the finished dosage form is related to a certain extent to the minimal inhibitory concentration of active substance against the infection causative agents and its particular type of formulation.
• the dosage may obviously be adjusted according to the severity of the infection, the type, age and conditions of the patient, the formulation selected for the administration, the administration schedule, etc.
• Experimental tests for determining the sensitivity of the microorganisms isolated from the patient may also offer useful indication to select the appropriate dosage.
• An effective dosage may be in general in the range 0.5-100 mg/kg body weight/day, preferably 5-50 mg/kg body weight/day.
• the prescribing physician will be able to determine the optimal dosage for a given patient in a given situation.
• the antibiotic compounds of the invention may be administered by parenteral (intramuscular, intravenous, subcutaneous, etc.) or oral route; the nature of the compound will determine the specific route of administration that may be employed.
• parenteral intramuscular, intravenous, subcutaneous, etc.
• oral route is generally preferred; otherwise, and also in the case of the unconscious or uncooperative state of the patient, parenteral administration of the active substance may conveniently be employed.
• the compounds of the invention can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, powder, solutions, suspensions or emulsions.
• the solid unit dosage form can be a capsule of the ordinary gelatin type containing lubricants and inert filler, such as lactose, sucrose and cornstarch.
• the compounds of general formula I can be tableted with conventional tablet bases such as lactose, sucrose and cornstarch in combination with binders, such as acacia, cornstarch or gelatin, disintegrating agents such as potato starch or alginic acid and a lubricant such as stearic acid or magnesium stearate.
• a unit dosage for oral administration may contain, for instance, from 50 to 700 mg of the active ingredient, preferably about 150 to 500 mg of the active ingredient.
• the compounds of the invention may be formulated into suitable injectable preparations containing a liquid vehicle.
• a liquid vehicle normally has no therapeutic effect and should not be toxic.
• suitable vehicles for preparing injectable dosage forms of the compounds of the invention are water, aqueous vehicles (e.g. Sodium chloride injections, Dextrose injections, etc.), water miscible solvents (e.g. ethyl alcohol, polyethylene glycol, propylene glycol, etc.) and non-aqueous vehicles (e.g. "fixed oils” such as corn oil, cottonseed oil, peanut oil and sesame oil).
• the injectable preparation may further contain buffers for stabilizing the solution (e.g. citrates, acetates and phosphates) and/or antioxidants (e.g. ascorbic acid or sodium bisulfite).
• the nature of the product will determine the specific route of administration (e.g. intramuscular, intravenous or subcutaneous) that may be employed.
• the desired route of administration will place requirements on the formulation. For example, suspensions would not be administered directly in the blood stream because of the danger of insoluble particles blocking capillaries, whilst solutions to be administered subcutaneously would require strict attention to tonicity adjustment, otherwise irritation of the nerve endings in the anatomical area would give rise to pronounced pain.
• the compounds of the invention may also be used as animal growth promoters.
• a compound of the invention is administered orally in a suitable feed.
• concentration employed will reflect the amount of active agent needed for a growth promotant effect and the amount of feed normally consumed.
• the addition of the active compound of the invention to animal feed is preferably accomplished by preparing an appropriate feed premix containing the active compound in an efficacious amount and incorporating the premix into the complete ration.
• an intermediate concentrate or feed supplement containing the active ingredient can be blended into the feed.
• Preparations A to G refer to the preparation of the starting materials (the compounds obtained are marked with letter P, i.e. P1 to P7);
• Examples A to I refer to the preparation of intermediate compounds (the compounds obtained are marked with letter E, i.e. E1 to E9);
• Examples 1 to 30 refer to the preparation of the compounds of formula I, and also of those compounds wherein R is (C 1 -C 8 )alkyl (the compounds obtained are marked with letter C, i.e. C1 to C30).
• reaction is followed by means of TLC plates (silica-gel 60 F 254 pre-coated, 5 ⁇ 10 cm, thickness 0.25,Merck); the purification of the products obtained by flash-chromatography is performed on silica gel (32-63, 60 ⁇ ;
• N-Bromosuccinimide (2.2 g) in dimethylformamide (5 ml) is slowly dropped into a cold (5° C.) solution of Compound P2 (7.7 g) and triethylamine (1.25 g) in dimethylformamide (25 ml).
• the reaction mixture is stirred for 3 hours at room temperature, then 1,1-diethylthiourea (1.8 g) in dimethylformamide (4 ml) is added. Stirring is continued for 1.5 h, then ascorbic acid (2.6 g) in water (5 ml) is added.
• the mixture is allowed to rest overnight and then is poured into water (300 ml) and extracted with ethyl acetate (3 ⁇ 100 ml).
• ethyl 4,6-dichloronicotinate (17 g) is boiled for 4 hours with 24% sulfuric acid (400 ml).
• the white crystals that separate are filtered from the boiling solution and 4-chloro-6-hydroxynicotinic acid (6 g, m.p. 299°-300° C.) is obtained; the acidic solution is left overnight in the refrigerator.
• the crystallized solid is collected by filtration and dried under vacuum, thus obtaining 6-chloro-4-hydroxynicotinic acid (4.6 g) m.p. 231°-233° C.
• the alkaline solution is concentrated to small volume, cooled and acidified to pH 4 with 1N hydrochloric acid.
• the white material that precipitates is collected and crystallized from ethanol to give 6-chloro-1,4-dihydro-1-ethyl-4-oxo-3-pyridine-carboxylic acid (3 g) m.p. 155°-57° C.
• Preparation F Potassium salt of 1-ethyl-1,4-dihydro-6-(4-methyl-1-piperazinyl)-4-oxo-3-pyridinecarboxylic acid (Compound P6)
• the potassium salt of compound P4 is prepared by adding 1N potassium hydroxide (6 ml) to the free acid dihydrochloride (676 mg-2 mmol) dissolved in water (5 ml). The aqueous solution is evaporated to dryness and the residue treated with boiling ethanol (20 ml). The insoluble material (KCl) is filtered and the alcoholic solution re-evaporated to dryness. The solid residue is triturated with diethyl ether, collected by filtration and dried over phosphorous pentoxide under vacuum giving the desired potassium salt (575 mg).
• the potassium salt of compound P5 is prepared by adding 1N potassium hydroxide (2 ml) to the free acid (705 mg-2mmol) dissolved in ethanol (10 ml). The resulting solution is evaporated to dryness and the solid residue is triturated with a 1:1 mixture of diethyl ether/ethanol, collected by filtration and dried over phosphorous pentoxide under vacuum giving the desired potassium salt (672 mg).
• N-methylen-t-butylamine (1.92 g) is slowly dropped into a cooled solution (15° C.) of Compound C1 (3.3 g) in tetrahydrofuran (25 ml). After an addition of tert-butylamine (0.4 ml), the reaction mixture is stirred for 5 minutes, then manganese dioxide (1.7 g) is added and stirring continued overnight at 48° C. After cooling, the mixture is filtered and dropped into a cold solution (0° C.) of 16% sulfuric acid (15 ml), ascorbic acid (3 g) and tetrahydrofuran (5 ml).
• step a By following the procedure of Example 17, step a, but starting from Compound C3 (4.1 g), N-methylen-t-butylamine (2.6 g), tert-butylamine (0.5 ml) and manganese dioxide (2.3 g), 36-chloro-formylrifamycin SV is obtained (3.9 g).
• step b By following the procedure of Example 17, step b, but starting from the above obtained compound (3.9 g) and 1-Amino-4-methylpiperazine (0.67 g), the title compound (2.92 g) is obtained.
• Example 25 starting from Compound P7 (500 mg) and Compound C19 (500 mg), the pure title compound (420 mg) is obtained.

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• 1994
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